CLONING THE BREAKPOINT CLUSTER REGION OF THE INV(16) IN ACUTE NONLYMPHOCYTIC LEUKEMIA M4 EO

The pericentric inversion of chromosome 16 and the t(16;16) are two re current aberrations in bone marrow of patients with acute nonlymphocyt ic leukemia subtype M4 Eo, characterized by abnormal eosinophilic gran ulation. We describe here the precise localization of the breakpoints using fluorescence in situ hybridization (FISH) with cosmids spread ov er the short arm of chromosome 16 and the detection, isolation and cha racterization of a 14Kb EcoRI fragment containing a cluster of breakpo ints. First, cosmids were mapped to intervals defined by constitutiona l 16p rearrangements, second, the inv(16) and t(16;16) breakpoints wer e mapped to one of the intervals using FISH with the mapped cosmids an d third, cosmids within this interval were ordered using two color int erphase FISH. An STS of the cosmid closest to the breakpoints was then used to isolate five YACs, which did span all of the 16 inv(16) break points and one t(16;16) breakpoint analysed. In the DNA of one inv(16) patient we detected an additional submicroscopic deletion immediately proximal to the 16p breakpoint. Since this patient has the same pheno type, the 16p sequences proximal to the breakpoint seem non-essential to M4 Eo. This implies that the pathologic event is the juxtaposition of sequences distal to the 16p breakpoint with sequences proximal to t he 16q breakpoint. While four of the five YACs showed instability of t he region around the inv(16) breakpoint, DNA halo analysis allowed us to identity one YAC which was co-linear with normal genomic DNA and ha s yielded the actual breakpoint sequences which could be subcloned int o cosmids and fosmids. The breakpoints of five patients clustering in the close vincinity of a BgIII site in a 14Kb EcoRI fragment. This nar row clustering is a strong indication that the M4 Eo specific sequence s are localized very nearby or at the break points. This region is now under further investigation.